Team:Paris Bettencourt/Protocols
Ferment It Yourself
iGEM Paris-Bettencourt 2O15
- Background
- Design
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Protocols
Analytical digestion protocol
- Prepare the following mix:
- 2µL 10X Digestion buffer
- 0.5µL Eco31I
- 0.5µL BbsI
- 2µL of DNA (200ng)
- 15µL water
- incubate 1h at 37°C
Annealing Protocol
- Phosphorylation of the oligos
- 5.6μL DNAse/RNAse free water
- 6.0μL oligo 1 (10µM)
- 6.0μL oligo 2 (10µM)
- 2.0μL 10X T4 DNA ligase buffer
- 0.4μL T4 PolyNucleotide Kinase Total: 20μL
- incubate 30min at 37°C
- add 1μL of 1M NaCl
- incube 5min at 95°C
- let the mix cool down
- use 2μL of the mix as a 10X solution
Chemical test for competent cells
- 20-30 sec at 8-10 krpm for the DNA tube (from the kit).
- Thow cell competent on ice. Label 2 ml eppendorf µtube for each concentration and put it on ice.
- Add 1µl of DNA into each µtube.
- Add 50µl of competent cell into each tube. Flick gently to mix. Incubate in ice for 30 minutes. Pre-heat waterbath at 42°C.
- Heat-shock cell by placing in waterbath 1 minute. CAUTION : The top need to be close to the water level, but not immerge in.
- Keep back the tube to ice for 5 minutes.
- Add 200µl of SOC media, incubate 37°C for 2h. Prepare agar plate of the media that you want, and label it (3 plate for each concentration).
- Add 20µl of each tube n the appropriate plate. Incubate 37°C for OVN (12-16h).
Electroporation
- Thaw electrocompetent cells on ice
- Add 2µL of ligation product or 0.5µL of native plasmid to the cells
- Transfer the cells in an 0.2mm electroporation cuvette
- put the cuvette in the electroporation device and pulse the cells at 2.5kV, 200 Ohms and 25µF
- add 200µL of LB right after pulsing
- recover 2 hours at 37°C
- plate 200µL and 50µL of the cells on LB + erythromycin (200µg/mL), incubate overnight at 37°C
Electrocompetent Cells
- Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α
- Incubate until the the DO600 reach 0.5 to 0.7
- Place the cultures on ice for 15 minutes
- For the culture in cold sterile 50mL falcon tubes
- Centrifuge them for 10 minutes at 6000rpm
- Throw the supernatant
- Resuspend the cells in 50mL cold distilled water
- Centrifuge them for 10 minutes at 6000rpm
- Throw the supernatant
- Resuspend the cells in 25mL cold distilled water
- Centrifuge them for 10 minutes at 6000rpm
- Throw the supernatant
- Resuspend the cells in 12.5mL cold 10% glycerol
- Centrifuge them for 10 minutes at 6000rpm
- Throw the supernatant
- Resuspend the cells in 5mL cold 10% glycerol
- Make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C
- Prepare the following mix: